This research proposal deals with the kinetics of inhibition, by vanadate ion, of the membrane transport enzyme, sodium and potassium ion activated adenosine triphosphatase (NaK-ATPase). This enzyme mediates the active transport of Na and K in all living cells. Vanadate (the plus-5 oxidation state of vanadium) is a trace element of natural occurrence in living systems. It is present in cells at concentrations which might inhibit NaK-ATPase in vivo, and it might function as an endogenous regulator of NaK-ATPase. Inhibition of NaK-ATPase by vanadate is markedly influenced by all the ligands which are required for enzyme activity (Mg, K, Na and ATP). The overall objective of this proposal is to study the mechanism of inhibition of NaK-ATPase by vanadate and by those ligands which influence this inhibition. We will study the effects of vanadate and ligand concentrations on the following activities: (1) NaK-ATPase, (2) K-dependent phosphatase activity catalyzed by NaK-ATPase, and (3) Na-dependent ATPase activity catalyzed by NaK-ATPase. We will also study the effects of vanadate on the turnover of the phosphoenzyme intermediate of NaK-ATPase. Finally, we will compare the sensitivities to inhibition by vanadate of NaK-ATPase preparations from various sources. The methods include standard kinetic analysis of enzyme reactions, and in some experiments, phosphorylation and de-phosphorylation will be measured directly with terminally labeled 32P-ATP as the substrate. We intend to use vanadate as a tool to further examine the interactions between the binding sites for natural ligands of NaK-ATPase. This study will provide information on the regulation of ATP hydrolysis and cation transport by NaK-ATPase.